Oncogenic KRAS Occurs after Prolonged In Vitro Arsenite Exposure of Human Prostate Epithelial Cells

Inorganic arsenic is an environmental human carcinogen of the urinary tract that includes the prostate gland, which may be among its many target organs. The CAsE-PE cell line was derived from prolonged (29 weeks), continuous, in vitro exposure of the normal human immortalized RWPE-1 prostate epithelial cells to 5uM arsenite (As). CAsE-PE cells become malignantly transformed according to the above three criteria but the mechanism remains unclear. We performed RNA-seq for differential gene expression and targeted sequencing to gain insight into in vitro As transformation. RNA-seq showed >7,000 significantly altered (>2-fold) transcripts in CAsE-PE cells compared to RWPE-1. Notably, KRAS was increased over 400-fold and PSA (KLK3) rose over 50-fold. Pathway analysis of altered transcripts supported increased cell growth, cell motility and survival pathways in CAsE-PE cells. Connectivity analysis showed 34 annotated KRAS downstream transcripts were increased more than 5-fold, as well as an up-regulation of several growth factors. Targeted DNA sequencing of the KRAS gene revealed an allelic imbalance with high expression of a mutated transcript carrying an oncogenic mutation at codon 12 accompanied by silent mutations and low wt allele expression. Parallel cultures of RWPE-1 cells remained WT KRAS genotype. qPCR showed increased number of KRAS gene copies and additional work found KRAS-4b as the predominant transcript variant compared to more KRAS-4a in RWPE-1 cells. These data are consistent with KRAS driven proliferation pathways found in spontaneous tumors and transformed cell lines. Increased expression and mutation of KRAS appears to explain As-induced transformation in this in vitro model system.